Substrate specificity of lysophospholipase D which produces bioactive lysophosphatidic acids in rat plasma

Previously we reported that lysophospholipase D in rat plasma hydrolyzes endogenous unsaturated lysophosphatidylcholines (LPCs) preferentially to saturated LPCs to lysophosphatidic acids with growth factor-like and hormone-like activities. In this study, we examined the possibility that association of LPCs with different proteins in rat plasma has an effect on the preference of lysophospholipase D for unsaturated LPCs. Large portions of various LPCs were found to be recovered in the lipoprotein-poor bottom fraction. Furthermore, the percentages of LPCs associated with albumin isolated from rat plasma were shown not to be consistent with their percentage conversions to lysophosphatidic acids by lysophospholipase D on incubation of rat plasma at 37 degrees C. These results indicate that distinct distributions of LPCs in the plasma protein fractions are not critical factors for the substrate specificity of lysophospholipase D. Experiments with Nagase analbuminemic rats suggested that albumin-LPC complexes are not necessarily required for the hydrolysis by lysophospholipase D; lipoprotein-associate LPCs appeared to be good substrates for the phospholipase. We found that both saturated and unsaturated LPCs are present mainly as 1-acyl isomers in rat plasma. This result indicates that the preference of lysophospholipase D for unsaturated LPCs is not attributable to a difference in position of the acyl group attached to the glycerol backbone of LPC. In addition, lysophospholipase D was also found to attack choline phospholipids with a long chain group and a short chain alkyl group, although their percentage hydrolyses were low. Taken altogether, these results suggest that lysophospholipase D shows higher affinities for free forms of unsaturated acyl type LPCs equilibrated with albumin-bound and lipoprotein-associated forms, than for free forms of saturated acyl type LPCs and analogs of platelet-activating factor.


Metal-ion stimulation and inhibition of lysophospholipase D which generates bioactive lysophosphatidic acid in rat plasma

We found that lysophospholipase D (LPLD) in rat plasma prefers unsaturated to saturated lysophosphatidylcholines as substrates, generating a biologically active lipid, lysophosphatidic acid, but it does not hydrolyze diacyl-phospholipids. In this study, this LPLD required a metal ion for activity, Co2+ being the most effective, followed in order by Zn2+, Mn2+, and Ni2+. This metal-ion-stimulated LPLD with unique substrate specificity, which has not been described previously, was susceptible to thiol-blocking reagents and serine esterase inhibitors, but not to a histidine-modifying reagent. Consistent with results using thiol-modifying agents, short-chain fatty aldehydes, secondary products of lipid peroxidation, were found to inhibit LPLD. Addition of dibutylhydroxytoluene or butylhydroxyanisole to the plasma increased the activity of this enzyme, probably in a manner independent of its antioxidant activity, since another antioxidant, propyl gallate, was rather inhibitory. These results suggest that rat plasma contains an active LPLD that differs in some properties from other members of the known phospholipase D family detected in animal tissues and body fluids.


Rate constants for quenching singlet oxygen and activities for inhibiting lipid peroxidation of carotenoids and alpha-tocopherol in liposomes

The (1)O2 quenching rate constants (kQ) of alpha-tocopherol (alpha-Toc) and carotenoids such as beta-carotene, astaxanthin, canthaxanthin, and lycopene in liposomes were determined in light of the localization of their active sites in membranes and the micropolarity of the membrane regions, and compared with those in ethanol solution. The activities of alpha-Toc and carotenoids in inhibiting (1)O2-dependent lipid peroxidation (reciprocal of the concentration required for 50% inhibition of lipid peroxidation: [IC50](-1)) were also measured in liposomes and ethanol solution and compared with their kQ values. The kQ and [IC50](-1) values were also compared in two photosensitizing systems containing Rose bengal (RB) and pyrenedodecanoic acid (PDA), respectively, which generate (1)O2 at different sites in membranes. The kQ values of alpha-Toc were 2.9 x 10(8) M(-1) s(-1) in ethanol solution and 1.4 x 10(7) M(-1) s(-1) (RB system) or 2.5 x 10(6) M(-1) s(-1) (PDA system) in liposomes. The relative [IC50](-1) value of alpha-Toc in liposomes was also five times higher in the RB system than in the PDA-system. In consideration of the local concentration of the OH-group of alpha-Toc in membranes, the kQ value of alpha-Toc in liposomes was recalculated as 3.3 x 10(6) M(-1) s(-1) in both the RB and PDA systems. The kQ values of all the carotenoids tested in two photosensitizing systems were almost the same. The kQ value of alpha-Toc in liposomes was 88 times less than in ethanol solution, but those of carotenoids in liposomes were 600-1200 times less than those in ethanol solution. The [IC50](-1) value of alpha-Toc in liposomes was 19 times less than that in ethanol solution, whereas those of carotenoids in liposomes were 60-170 times less those in ethanol solution. There were no great differences (less than twice) in the kQ and [IC50](-1) values of any carotenoids. The kQ values of all carotenoids were 40-80 times higher than that of alpha-Toc in ethanol solution but only six times higher that of alpha-Toc in liposomes. The [IC50](-1) values of carotenoid were also higher than that of alpha-Toc in ethanol solution than in liposomes, and these correlated well with the kQ values.


Accumulation of VariousN-Acylethanolamines IncludingN-Arachidonoylethanolamine (Anandamide) in Cadmium Chloride-Administered Rat Testis

Changes in the levels of various molecular species of N-acylethanolamine in CdCl2-administered rat testis were examined. We found that the levels of various N-acylethanolamines including anandamide (N-arachidonoylethanolamine), an endogenous cannabinoid receptor ligand, were dramatically increased in CdCl2-admin-istered rat testis. Such changes were particularlyprominent for saturated and monoenoic species such as N-palmitoyl species (39-fold at 9 h) and N-stearoyl species (21-fold at 9 h), compared with unsaturated fatty acid-containing species such as anandamide (5-fold at 9 h). Noticeably, increased levels were observed of not only N-acylethanolamines but also several species of N-acylphosphatidylethanolamine, potential precursors for N-acylethanolamines. We confirmed that the rat testis microsomal fraction contains phosphodiesterase activity catalyzing the release of N-acylethanolamine from N-acylphosphatidylethanolamine and transacylase activity catalyzing the formation of N-acylphosphatidylethanolamine from phosphatidylethanolamine and phosphatidylcholine. These enzyme activities were not dramatically different in the microsomal fraction obtained from CdCl2-administered rat testis compared with that in the case of control rat testis, at least when estimated in cell-free assay systems, suggesting that the accessibility of the substrates to the enzymes may be increased in CdCl2-administered rat testis to generate a large amount of N-acylethanolamine. Possible pathophysiological implications of the augmented generation of N-acylethanolamine including anandamide in CdCl2-administered rat testis were discussed.


2-Arachidonoylglycerol, an endogenous cannabinoid receptor agonist: identification as one of the major species of monoacylglycerols in various rat tissues, and evidence for its generation through Ca2+-dependent and -independent mechanisms


Undifferentiated HL-60 cells internalize an antitumor alkyl ether phospholipid more rapidly than resistant K562 cells

In this study, we confirmed a previous finding that 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (methyl-PAF) expresses higher antineoplastic activity against the promyelocytic leukemia cell line HL-60, than against the erythroleukemic cell line K562, and intended to clarify the reason for this. Using an albumin back-exchange method, we measured the rates of binding and internalization of [3H]methyl-PAF by HL-60 and K562 cells. We found that methyl-PAF associated very rapidly and to similar extents with the two types of cells at low concentrations of extracellular bovine serum albumin, but that when bound to the cell surface, it was internalized into HL-60 cells faster than into K562 cells. The internalization of methyl-PAF by HL-60 cells was concentration-independent, intracellular ATP-independent and susceptible to thiol group-modifying reagents and cytochalasin B. Thus the inward transbilayer movement of methyl-PAF seems to occur by cytochalasin B-sensitive protein-mediated mechanism based on passive diffusion not requiring energy, in which SH-groups of protein play a critical role. We also found that the internalization of 1-hexadecanoyl-2-(4,4-difluoro-5,7- dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphocholine (Bodipy-C5-PC), whose structure resembles that of methyl-PAF, into HL-60 cells was faster than that into K562 cells. Using a combination of an albumin back-exchange method and observation by confocal laser scanning microscopy, we next examined the intracellular distribution of this fluorescent phospholipid probe after its internalization. Intracellular membranes, especially those peripheral to nuclei, were fluorescence-labeled in both HL-60 and K562 cells, but fluorescence of the nuclear membranes was weak, suggesting that this probe seems mainly to accumulate in intracellular granules, and may interact directly with several key enzymes for phospholipid metabolism, leading to cell injury. Because the difference between the internalization rates of methyl-PAF in HL-60 and K562 cells was correlated with their different susceptibilities to the cytotoxic effect of methyl-PAF, we suggest that the capacities for uptake of methyl-PAF and its accumulation in intracellular membranes are critical factor for its induction of apoptosis. (c) 1998 Elsevier Science B.V.

Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism


Positive and negative controls by protein kinases of sodium-dependent Ca2+ efflux from cultured bovine adrenal chromaffin cells stimulated by lysophosphatidic acid

We previously found that lysophosphatidic acid (LPA), a bioactive phospholipid, induced Na+-dependent Ca2+ efflux from cultured bovine adrenal chromaffin cells, possibly by activating a Na+/Ca2+ exchanger. The present study on the structure-activity relationship of its action revealed that 1-acyl type LPAs were stronger stimulants than the corresponding 1-O-alkyl type LPAs having a long alkyl moiety with the same chain length. Lysophosphatidylglycerol, suramin and N-palmitoyl-tyrosine phosphoric acid have all been reported to inhibit the action of LPA in some animal cells and platelets, but only lysophosphatidylglycerol was found to inhibit selectively LPA-induced Ca2+ efflux from chromaffin cells. LPA-induced Ca2+ extrusion was suggested to be involved in both acceleration of return of intracellular Ca2+ in Fura 2-loaded bovine chromaffin cells after addition of carbachol, and inhibition of carbachol-induced catecholamine release when the cells were co-incubated with LPA. The Ca2+ efflux from chromaffin cells stimulated by LPA was augmented by their pretreatment with staurosporine or calphostin C, inhibitors of protein kinase C, but reduced by their preincubation with phorbol 12-myristate 13-acetate. Furthermore, the response to LPA was potentiated by sodium vanadate, a protein tyrosine phosphatase inhibitor, but inhibited by genistein, an inhibitor of protein tyrosine kinase. These results suggest that protein kinase C and protein tyrosine kinase are involved negatively and positively, respectively, in the signal transduction triggered by LPA, leading to activation of the Na+/Ca2+ exchanger.


Singlet oxygen scavenging by lpha-tocopherol and eta-carotene: Kinetic studies in phospholipid membranes and ethanol solution

The rate constants (ks) of 1O2 scavenging for alpha-tocopherol (alpha-Toc) and beta-carotene (beta-Car) were measured in liposome membranes, and compared with those in EtOH solution. 1O2 was site-specifically generated by photoirradiation using two photosensitizers, water-soluble Rose bengal (RB) and lipid-soluble 12-(1-pyrene)-dodecanoic acid (PDA). The ks value for beta-Car in EtOH solution was 1.3 x 10(10) M-1 s-1, which was 36 times that for alpha-Toc (3.6 x 10(8) M-1 s-1), but there was no difference between their ks values in liposomes (1.8 x 10(7) M-1 s-1 for beta-Car and 1.2 x 10(7) M-1 s-1 for alpha-Toc). In the liposomes, the ks value for alpha-Toc was affected by the membrane site where 1O2 was generated, which depended on the localization of the photosensitizer, being high at the membrane surface in the RB-system and low in the inner region of the membrane in the PDA-system. In contrast, the ks value for beta-Car was not affected by the 1O2-generating site. These differences were supposed to be caused by differences in the relative concentrations of 1O2 and active sites of alpha-Toc and beta-Car in the membranes. alpha-Toc and beta-Car inhibited 1O2-dependent peroxidation of egg yolk phosphatidylcholine (egg PC). The concentrations of alpha-Toc required for 50% inhibition of lipid peroxidation (IC50) were higher than those of beta-Car, being more than 6 times higher in EtOH solution and less than 2 times higher in liposomes. The ratio of the antioxidant activity of beta-Car to that of alpha-Toc was more in EtOH solution than in liposomes, and was well correlated with the ratio of their 1O2 scavenging rate constants.


Exogenously added alkylmethylglycerophosphocholine and alkylmethylcarbamylglycerophosphocholine accumulate in plasma membranes more than in intracellular membranes of rabbit platelets

We found that extracellular addition of 2% bovine serum albumin (BSA) to a suspension of rabbit platelets after stimulation with platelet-activating factor resulted in a biphasic extraction of [3H]1-O-alkyl-2-O-methyl (or 2-O-methylcarbamyl)-sn-glycero-3-phosphocholine. A fast phase of extraction of the phospholipid probe by BSA was found to be mainly due to removal of the probe remaining in an outer layer of platelet plasma membrane, whereas a second phase of extraction of the probe by BSA was mostly attributed to redistribution of the probe which had been flipped across the plasma membrane. On the basis of analysis of the biphasic extraction by BSA of 1-O-alkyl-2-O-methyl (or methylcarbamyl)-sn-glycero-3-phosphocholine at various times after its addition, we suggested that the radioactive phospholipid accumulated in plasma membrane more than in intracellular membranes of rabbit platelets. In similar experiments with guinea-pig polymorphonuclear leukocytes, we observed a monophasic extraction of 1-O-alkyl-2-O-methyl (or methylcarbamyl)-sn-glycero-3-phosphocholine by BSA, indicating its unidirectional movement across the plasma membrane.


Exogenous phosphatidic acid with saturated short-chain fatty acyl groups induces superoxide anion release from guinea pig peritoneal polymorphonuclear leukocytes by three different mechanisms

Treatment of suspensions of guinea pig peritoneal polymorphonuclear leukocytes (PMN) with four species of phosphatidate (PA) containing short-chain fatty acids induced sustained superoxide anion (O2-) production after a lag time. The rank order of efficiency of these PAs in triggering O2- production was PA8:0 [1,2-dioctanoyl-sn-glycerol-3-phosphate (GP)] > PA10:0 (1,2-didecanoyl-GP) > PA6:0 (1,2-dicaproyl-GP) > > PA12:0 (1,2-dilauroyl-GP). The O2- release from PMN stimulated with PA10:0 or PA12:0, but not with PA6:0 or PA8:0, was lowered by the addition of 1 mM extracellular Ca2+. Studies with various inhibitors showed that the mechanism of multiphasic O2- production induced by PA8:0 depended on its concentration: 1 and 3 microM PA8:0 induced O2- production constantly after a lag time through a protein kinase-dependent mechanism that was inhibited by 100 nM staurosporine. With concentrations of PA of 10 microM or more, an additional mechanism that was independent of protein kinase became operative and predominant over the protein kinase-dependent one. This protein kinase-independent mechanism was inhibited selectively by 80 microM TMB-8. Concentrations of 30, 60 and 100 microM PA first elicited transient O2- production via another protein kinase-dependent mechanism that was more sensitive to H-7 than to staurosporine, and then sustained O2- production, mainly driven by the protein kinase-independent mechanism. Metabolism of exogenously added [14C]PA8:0 in intact PMN was examined in the presence and absence of propranolol. Results suggest that PA itself is more important rather than its degradation products such as diacylglycerol, in inducing O2- production via three different mechanisms described above.


Kinetics and Dynamics of Singlet Oxygen Scavenging by -Tocopherol in Phospholipid Model Membranes

Scavenging of singlet oxygen (1O2) by alpha-tocopherol (alpha-Toc) was investigated in liposomes. 1O2 was generated by photoirradiation in the presence of two photosensitizers, water-soluble methylene blue (MB) and lipid-soluble 12-(1-pyrene)dodecanoic acid (PDA). The rates of oxidation of alpha-Toc differed depending on the photosensitizing dye and the membrane charge: in the MB-system, alpha-Toc was oxidized fast in negatively charged dimyristoylphosphatidylcholine (DMPC) liposomes containing dicetylphosphate (DCP) and slowly in neutrally charged DMPC liposomes and positively charged DMPC liposomes containing stearylamine (SA), but in the PDA-system, the oxidation rate was independent of the membrane charge. The charge-dependent difference in the MB-system would be due to the site of 1O2 generation depending on the charge-dependent distribution of MB, because positively charged MB increased the zeta-potential of DCP-DMPC liposomes by its interaction with DCP at the membrane surface, but changed the zeta-potentials of DMPC and SA-DMPC liposomes less because of its location in the bulk water phase. The oxidation rate of alpha-Toc in liposomes was different from that in EtOH solution: in the MB system, the oxidation rate was faster in EtOH solution than in DMPC or SA-DMPC liposomes but the same as that in DCP-DMPC liposomes. However, in the PDA system, the oxidation rate was slower in EtOH solution than in DMPC liposomes with or without a charge. Membrane fluidity changed the rate of alpha-Toc oxidation in liposomes, the rate being higher in the liquid crystalline phase than the gel phase, as judged by the higher rate in DMPC liposomes than in dipalmitoylphosphatidylcholine (DPPC) liposomes at 30 degrees C. The rate constants of alpha-Toc for scavenging, the chemical reaction and physical quenching of 1O2 were determined in membranes using DCP-DMPC liposomes labeled with 1,3-diphenyl-isobenzofuran (DPBF), which traps 1O2. These constants differed in the two photosensitizing systems, being higher in the MB-system than in the PDA-system, and were lower than those in EtOH solution.


Lipid peroxidation in low density lipoproteins from human plasma and egg yolk promotes accumulation of 1-acyl analogues of platelet-activating factor-like lipids

Oxidative modification of low density lipoprotein (LDL) is known to be a key event for induction of atherosclerosis. However, there has been little progress in structural elucidation of oxidized lipids, especially oxidatively fragmented phospholipids retaining a glycerol backbone. In this study, we found that LDL derived from egg yolk has no platelet-activating factor (PAF) acetylhydrolase activity, and that prolonged incubation of egg yolk LDL with Cu2+ resulted in the formation of various PAF-like lipids: 1-acyl type phosphatidylcholines with an sn-2-short-chain dicarboxylate or monocarboxylate group. Only a very small amount of the PAF-like lipid having an sn-2-short-chain monocarboxylate group was detected by gas chromatography-mass spectrometry in Cu(2+)-oxidized LDL from human plasma with high PAF-acetylhydrolase activity, which has been reported to hydrolyze PAF-like lipids to lysophosphatidyl-cholines. Preincubation of plasma LDL with diisopropyl fluorophosphate dose-dependently inhibited PAF-acetylhydrolase activity, resulting in accumulation of the PAF-like lipids when the LDL was oxidized with Cu2+. As well as PAF and lysophosphatidylcholines, several PAF-like lipids were found to inhibit [3H]thymidine incorporation into cultured vascular smooth muscle cells derived from rat aorta. The possible formation of PAF-like lipids by lipid peroxidation in LDL is discussed as well as its possible significance for induction of atherosclerosis.


Two effects of lysophosphatidic acid on Ca(2+)-movement in cultured bovine adrenal chromaffin cells

We found that lysophosphatidic acid (LPA) exerted two effects on Ca(2+)-movement in cultured bovine adrenal chromaffin cells. At concentrations of above 10(-5) M, it induced slight, but significant 45Ca2+ influx, resulting in release of a small portion of stored catecholamine. At high concentrations it also significantly increased the intracellular Ca2+ concentration in Fura-2-loaded cells. At concentrations as low as 10(-7) M, it stimulated extracellular Na(+)-dependent 45Ca2+ efflux, possibly by increasing Na+/Ca2+ exchange. The maximal efflux of Ca2+ attained with 10(-5) M LPA was inhibited by tyrosine kinase inhibitors, but augmented by a protein kinase C inhibitor. These results suggest that LPA-induced Ca2+ efflux is controlled positively and negatively by mechanisms involving tyrosine kinase and protein kinase C, respectively.

Journal of Lipid Mediators and Cell Signalling


Lipid peroxidation in egg phosphatidylcholine liposomes: comparative studies on the induction systems Fe2+/ascorbate and Fe3+-chelates/xanthine-xanthine oxidase.

Two typical systems of lipid peroxidation in egg yolk phosphatidylcholine (egg PC) liposomes were compared: an enzymic system involving superoxide (O2) generated by xanthine (X), xanthine oxidase (XO) and Fe(3+)-chelates (Fe(3+)-ADP and Fe(3+)-EDTA), and a non-enzymic system involving ascorbic acid (ASA) and Fe2+. These two systems exhibited a different pH-dependence: the rate in the enzymic system was maximal at pH 8-8.5, whereas that in the non-enzymic system was high below pH 7.4 and low above pH 7.6. The rates of lipid peroxidation differed with the membrane charge, and this charge-dependent phenomenon differed in the two peroxidation systems: in the Fe(3+)-chelates/X-XO-system, the rate was slow in neutrally charged egg PC liposomes and rapid in egg PC liposomes containing negatively charged dicetylphosphate (DCP) or positively charged stearylamine (SA), whereas in the Fe2+/AsA-system, the rate was rapid in neutral egg PC liposomes but no lipid peroxidation occurred in egg PC liposomes charged with DCP or SA. The decomposition rate of the hydroperoxide of PC (PC-OOH) incorporated into dimyristoyl-phosphatidylcholine (DMPC) liposomes differed depending on the membrane charge in the two systems and this charge-dependence of the rates correlated well with that of the initiation rate of lipid peroxidation dependent on membrane charge. In the Fe2+/AsA-system, lipid peroxidation depended on the endogenous presence of PC-OOH, and the amounts of PC-OOH required for initiation of the reaction differed depending on the membrane charge. However, in the Fe(3+)-chelates/X-XO-system, lipid peroxidation occurred very slowly in the absence of PC-OOH, but rapidly in its presence.


Regulations by cyclic nucleotides and phorbol ester of calcium efflux from cultured bovine adrenal chromaffin cells.


Vasopressor effect of lysophosphatidic acid on spontaneously hypertensive rats and Wistar Kyoto rats

Intravenous injection of lysophosphatidic acid (LPA, 1-acyl-sn-glycerol-3-phosphate) into conscious, spontaneously hypertensive rats (SHR) promptly elicited hypertension in a dose-dependent manner, its effect being significantly higher than in conscious, age-matched Wistar Kyoto strain rats (WKY). There was, however, no difference between the potencies of LPA in raising the mean blood pressure of SHR and WKY anesthetized with pentobarbital. The releases of norepinephrine, angiotensin II, prostaglandin and leukotriene were found not to be involved in the vasopressor effect of LPA in SHR, although thromboxane seemed to be slightly related to the action of LPA.

Research Communications in Molecular Pathology and Pharmacology


Platelet-activating factor and its structural analogues in the earthworm Eisenia foetida

The earthworm Eisenia foetida was shown to contain large amounts of ether-containing phospholipids such as alkylacylglycerophosphocholine (61.3% of choline glycerophospholipids) and alkenylacylglycerophosphoethanolamine (66.0% of ethanolamine glycerophospholipids). We also found a substantial amount of ether-containing PAF-like lipid in this animal, its level being increased after the animal is injured. We showed evidence that this PAF-like lipid consists of PAF and PAF analogues containing short chain fatty acids other than acetic acid. Notably, a propionic acid-containing species but not PAF itself, is the most predominant species in this animal. We also confirmed that the earthworms contain enzyme activities involved in the synthesis of PAF and short chain fatty acid-containing PAF analogues. Interestingly, the acetyltransferase activity in earthworms is resistant to high concentrations of the substrate lysophospholipid. Thus, both the structure of the PAF-like lipid and the properties of the enzymes involved in the PAF-like lipid metabolism in the earthworms are somewhat different from those in mammalian tissues.

Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism


Platelet activating factor (PAF)-like phospholipids formed during peroxidation of phosphatidylcholines from different foodstuffs

Previously, we reported that induction of peroxidation of synthetic phosphatidylcholines (PCs) containing a polyunsaturated fatty acid by Fe(2+)-EDTA in the presence of ascorbate resulted in the formation of four types of PCs with an sn-2-oxidatively fragmented acyl group, which had platelet-aggregating activity due to interaction with platelet-activating factor (PAF) receptors. These PCs were compounds with a short-chain monocarboxylate, omega-hydroxymonocarboxylate, dicarboxylate, and dicarboxylate semialdehyde residue, respectively. In this study, we investigated the PAF-like lipids formed during peroxidation of PCs from hen egg yolk, salmon roe, sea urchin eggs, and krill in an FeSO4/EDTA/ascorbate system. The platelet-aggregating activities of these oxidized PCs were all inhibited by FR-900452, an antagonist of PAF. The activity of oxidized krill PC, which was equivalent of 89.8 +/- 8.8 pmol 16:0-PAF/mumol of starting PC, was about 5 times those of oxidized PCs from salmon roe and sea urchin eggs, and about 50 times that of oxidized hen egg yolk PC. The PAF-like phospholipids that had different combinations of long-chain alkyl or acyl groups with one of the above four types of short-chain acyl groups were identified by gas chromatography-mass spectrometry. The results indicated that foodstuffs that are rich in 1-O-alkyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine are potential sources of compounds with high PAF-like activity formed by deleterious lipid peroxidation.

Bioscience, Biotechnology, and Biochemistry


Protein kinase c-independent inhibition of the Ca2+-activated K+ channel by angiotensin II and endothelin-1

We previously reported that the Ca(2+)-activated K+ channel (KCa-channel) in cultured smooth muscle cells from porcine coronary artery was inhibited by protein kinase C (C-kinase). In this study, inhibition of the KCa-channel by receptor-mediated vascular contractile agonists, such as angiotensin II (ANG II) and endothelin-1 (ET-1), was investigated by the patch-clamp technique. In cell-attached patches, addition of ANG II (500 nM) or ET-1 (50 nM) to the bath inhibited the KCa-channel activated by the calcium ionophore A23187 (10-20 microM). Phorbol 12-myristate 13-acetate (PMA, 1 microM), a C-kinase activator, also decreased the open probability of the KCa-channel. The PMA-induced decrease in the open probability was reversed by subsequent application of staurosporine (1 nM), a C-kinase inhibitor, but the ANG II- and ET-1-induced decreases were not reversed by subsequent application of staurosporine (> 30 nM). Pretreatment of smooth muscle cells with 30 nM staurosporine, a protein kinase inhibitor, or 1 mM neomycin, an inhibitor of phospholipase C, also did not abolish the inhibition of the KCa-channel by ANG II. Furthermore, ANG II inhibited the KCa-channel in cells in which C-kinase was down-regulated. These results indicate that, in porcine coronary artery smooth muscle cells, ANG II and ET-1 inhibit the KCa-channel by a C-kinase-independent mechanism.


Dynamics of xanthine oxidase- and Fe^{3+}-ADP-dependent lipid peroxidation in negatively charged phospholipid vesicles

Superoxide (O2-)-dependent lipid peroxidation on addition of xanthine oxidase (XO) and Fe(3+)-ADP was induced in egg phosphatidylcholine (PC) liposomes containing dicetylphosphate (DCP), which are negatively charged like biological membranes, but not in uncharged egg PC liposomes. Positively charged Fe(3+)-ADP interacted more with negatively charged egg PC-DCP liposomes than with uncharged egg PC liposomes. The activities of Fe(3+)-chelates for initiating O(2-)-dependent lipid peroxidation were in the order Fe(3+)-ADP > Fe(3+)-citrate > Fe(3+)-oxalate = Fe(3+)-malonate > Fe(3+)-EDTA = 0. This order was the same as that for the reduction rates of these Fe(3+)-chelates to Fe(2+)-chelates by O(2-)-generated by XO. Lineweaver-Burk plots showed that the chelators inhibited XO by different mechanisms: uncompetitively by ADP and adenosine and non-competitively by organic acid chelators (citrate and oxalate) and EDTA. These results suggest that ADP interacts with XO in a manner different from the other chelators. Lipid peroxidation by XO-xanthine and Fe(3+)-ADP was induced in egg PC liposomes containing a trace (0.31-0.35 mol%) of peroxidized egg PC (PC-OOH), but not in PC-OOH-free liposomes of egg PC obtained by their pretreatment with triphenylphosphine. PC-OOH incorporated into dimyristoyl phosphatidylcholine (DMPC) liposomes was degraded on addition of both XO-xanthine and Fe(3+)-chelate, but not of either one alone. alpha-Tocopherol in DMPC liposomes was oxidized on addition of XO-xanthine and Fe(3+)-chelates in the presence, but not in the absence of PC-OOH. Furthermore, PC-OOH was required for decrease of the ESR spectrum of the spin probe 12-(N-oxyl-4,4'-dimethyloxazolidin-2-yl)stearic acid, which labels the hydrophobic region of egg PC liposome membranes, on addition of XO-xanthine and Fe(3+)-chelates. These results indicate that the "induction message of lipid peroxidation," which is associated with reduction of Fe(3+)-ADP by O2- and concurrent degradation of PC-OOH, must be transferred from the membrane surface to the inner hydrophobic region of the membranes.


Stimulatory effect of lysophosphatidic acid on calcium efflux from cultured bovine adrenal chromaffin cells


Regulation by cyclic nucleotides and phorbol ester of calcium efflux from cultured bovine Adrenal chromaffin ccells


Stimulatory effect of angiotensin II on calcium efflux from cultured bovine adrenal chromaffin cells

The effect of angiotensin II on Ca2+ mobilization in cultured bovine adrenal chromaffin cells was examined. Angiotensin II (10(-7)M) increased the intracellular free Ca2+ level ([Ca2+]i) to a peak in the presence or absence of extracellular Ca2+, followed by decrease with time. Angiotensin II (10(-9)-10(-6)M) also stimulated 45Ca2+ efflux from cultured bovine adrenal chromaffin cells in a concentration-dependent manner. Its stimulatory effect on 45Ca2+ efflux was inhibited by the angiotensin II antagonist [Sar1, Ile8]-angiotensin II or [Sar1, Val5, Ala8]-angiotensin II. The increase in angiotensin II-stimulated 45Ca2+ efflux was dependent on the extracellular Na+ concentration. Angiotensin II also increased 22Na+ influx into the cells. These results indicate that stimulation of the angiotensin II receptor induces extracellular Na(+)-dependent Ca2+ efflux from cultured bovine adrenal chromaffin cells, probably by acceleration of Na+/Ca2+ exchange.


Effect of lysophosphatidic acid on the preimplantation development of mouse embryos

The effect of lysophosphatidic acid (LPA) on the preimplantation development of mouse embryos was examined. The blastocyst rates from 2-cell and 4-cell stage embryos were significantly higher in the presence of LPA at the pronuclear stage. However, the blastocyst rate from 4-cell stage embryos was significantly lower when LPA was added to the medium at the 4-cell stage than when it was added at the pronuclear stage. Furthermore, pretreatment of pronuclear stage embryos with pertussis toxin suppressed LPA-induced embryo development, suggesting that it is probably mediated by a Gi-protein-linked receptor.


Lysophosphatidic acids induce proliferation of cultured vascular smooth muscle cells from rat aorta

Lysophosphatidic acids (LPA) with a C18 fatty acyl group accelerated thymidine incorporation into cultured rat aortic vascular smooth muscle cells and stimulated their cell division. LPA acted synergistically with epidermal growth factor and fibroblast growth factor but additively with platelet-derived growth factor. The stimulatory actions of LPA were suggested to be rather specific from the following findings: 1) their stimulation of DNA synthesis increased with an increase in their acyl moiety; 2) lysophosphatidylcholine, a neutral lysophospholipid, had no mitogenic action but was cytotoxic at high concentrations; and 3) LPA induced a rapid external Ca(2+)-independent increase in intracellular Ca2+ concentration ([Ca2+]i) in single fura 2-loaded cells that resembled the receptor-mediated increases in [Ca2+]i triggered by different agonists, whereas lysophosphatidylcholine provoked a slow sustained increase in [Ca2+]i in an external Ca(2+)-dependent manner. These results are discussed in relation to the possible pathophysiological role of LPA.


Biochemical Characterization of the Interaction of Lipid Phosphoric Acids with Human Platelets: Comparison with Platelet Activating Factor

A series of lipid phosphoric acids, including 1-O-alkyl-2-lyso-glycerophosphoric acid, 1-O-acyl-2-lyso-glycerophosphoric acid, hexadecylpropanediolphosphoric acid, N-acyl-2-aminoethanolphosphoric acid, sphingosine phosphoric acid, and certain homologues and analogues, were synthesized and characterized by thin-layer chromatography, fast-atom bombardment mass spectrometry, and their ability to aggregate human platelets. The presence of a receptor for these lipid phosphoric acids that is distinct from the PAF receptor is strongly suggested from experiments involving a desensitization procedure, platelet-activating factor (PAF) receptor antagonists, and inhibitors of the lipid phosphoric acids. The unique features of the interaction of these lipid phosphoric acids with platelets include: (a) evidence for a separate receptor(s) for this diverse group of synthetic compounds, (b) no requirement for stereospecificity (i.e., no glycerol backbone), and (c) a structural requirement for a long-chain hydrocarbon residue covalently bound to a phosphoric acid residue. In the interaction of these compounds with the platelet, it is mandatory that extracellular Ca2+ and ADP be present for maximum biological activity. The potential involvement of a lipid phosphoric acid receptor, which could form a component of the activation pathway associated with various lysophospholipids and analogues, such as PAF, via a phospholipase D activation, is discussed.


Platelet-aggregating effects of platelet-activating factor-like phospholipids formed by oxidation of phosphatidylcholines containing an sn-2-polyunsaturated fatty acyl group

Previously, we reported the formation of four kinds of phosphatidylcholines (PC) with a short-chain monocarboxylate, dicarboxylate, dicarboxylate semialdehyde or omega-hydroxymonocarboxylate group by oxidation of PCs containing polyunsaturated fatty acid (PUFA) in an FeSO4/ascorbate/EDTA system. In this study, we identified these novel phospholipids by GC-MS as oxidation products of two alkyl ether-linked PCs, 1-O-hexadecyl-2-docosahexaenoyl and 1-O-hexadecyl-2-arachidonoyl-sn-glycero-3- phosphocholine (GPC). The sn-2-acyl moieties of oxidatively fragmented PCs derived from PCs containing docosahexaenoate were one methylene unit shorter than those detected as major oxidation products of PCs containing arachidonate. The platelet-aggregations induced by the oxidized PCs were all inhibited by FR-900452, an antagonist of platelet activating factor (PAF). The PAF-like activity of oxidized 1-O-hexadecyl-2-docosahexaenoyl-GPC, which was equivalent of 1372 +/- 262 pmol 16:0-PAF/mumol starting PC, was 5 times that of oxidized 1-O-hexadecyl-2-arachidonoyl-GPC and 150 times that of oxidized 1-palmitoyl-2-docosahexaenoyl-GPC, suggesting that both an sn-1-alkyl ether linkage and an sn-2-acyl group with a short chain length are important structural requirements for induction of platelet aggregation. These possibilities were confirmed by experiments on the platelet-aggregating activities of synthetic PAF-like compounds. Quantitative measurements by GC-MS of PAF-like phospholipids formed by lipid peroxidation and the activities of synthetic PAF-like phospholipids, suggested that the activities of most oxidized PCs containing PUFA were ascribable to those of PCs with an sn-2-short-chain monocarboxylate group.

Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism


Platelet-aggregating effects of platelet-activating factor-like phospholipids formed by oxidation of phosphatidylcholines containing an sn-2 polyunsaturated fatty acyl group


Formation of platelet-activating factor-like phospholipids by Fe2+/ascorbate/EDTA-induced lipid peroxidation


Isolation of a phospholipid inhibitor of platelet activating factor-induced activity from perfused rat liver: identification as phosphatidylglycerol

An endogenous inhibitor of platelet activating factor action was isolated from perfused rat liver. It was purified by thin-layer chromatography and high-performance liquid chromatography and subjected to chemical modifications in order to identify its structure. On the basis of its fast atom bombardment-mass spectrum it was characterized as phosphatidylglycerol composed mainly of 16:0/18:1 and 16:0/20:2 fatty acyl chains ([M+H]+ at m/z 749 and 775, respectively) and very minor levels of 18:0/18:1 and 18:0/20:2. The purified compound exhibited inhibition on rabbit platelet aggregation induced by 5 x 10(-10) M platelet activating factor (PAF) at an EC50 value near 2.5 x 10(-6) M and on the serotonin secretion at an EC50 7 x 10(-6) M. Other phospholipids isolated from the liver preparations, such as phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cardiolipin (diphosphatidylglycerol), and phosphatidic acid, exhibited no inhibitory activity in the concentration range from 1 x 10(-4) to 1 x 10(-7) M nor did they induce any aggregation, or lysis, of the platelets. Of importance, phosphatidylglycerol could inhibit thrombin- and ADP-induced aggregation of rabbit platelets. These results suggested a possible site of inhibition common to the signal transduction pathway of these agonists. Preliminary binding experiments showed a noncompetitive type of inhibition on PAF binding to intact rabbit platelets.


Possible influence of lysophospholipase on the production of 1-acyl-2-acetylglycerophosphocholine in macrophages

The rate of production of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (acylPAF) was measured in macrophages following the incorporation of [3H]acetate. Upon activation by A23187, guinea pig alveolar macrophages incorporated [3H]acetate into PAF, but a little radioactivity was found in acylPAF. However, labeling of acylPAF and PAF with [3H]acetate was greatly enhanced in A23187-stimulated alveolar macrophages that had been pretreated with phenylmethanesulphonyl fluoride (PMSF). [3H]PAF was predominantly converted to 1-[3H]alkyl-2-acyl glycerophosphocholine, but [14C]acylPAF rapidly hydrolyzed to 14C-labeled free fatty acid by the incubation with lysates prepared from macrophages. The deacetylation of [14C]acylPAF and [3H]PAF by acetylhydrolase and also the hydrolysis of [14C]lysoPC by lysophospholipase were strongly inhibited in macrophages that had been pretreated with PMSF, while PMSF failed to inhibit the activities of acetyltransferase and acyltransferase. The relative proportions of PAF and acylPAF were quite different in different types of cells. In contrast to alveolar macrophages, peritoneal macrophages, neutrophils and spleen cells from guinea pigs incorporated 2-4 times more [3H]acetate into acylPAF than into PAF. The presence of high levels of acylPAF in peritoneal macrophages was confirmed by GLC-MS analysis. The activities of lysophospholipase, acetylhydrolase and acetyltransferase were measured in alveolar and peritoneal macrophages to determine whether the preferential formation of acylPAF as compared to PAF in peritoneal macrophages was due to differences in these activities between alveolar and peritoneal macrophages. The activity of acetylhydrolase of peritoneal macrophages was almost the same as that in alveolar macrophages. The activity of acetyltransferase in peritoneal macrophages was about half of that in alveolar macrophages. However, the activity of lysophospholipase in peritoneal macrophages was one-sixth of that in alveolar macrophages. These results suggest that lysophospholipase is one of the primary factors involved in the control of the production of acylPAF in activated cells, and that it acts by modulating the availability of lysoPC for the synthesis of acylPAF. Furthermore, high levels of activity of lysophospholipase allow the preferential formation of PAF, via the rapid hydrolysis of lysoPC which would act as a competitive inhibitor of the incorporation of acetate into lysoPAF.


Transbilayer movement and metabolic fate of ether-linked phosphatidic acid (1-O-Octadecyl-2-acetyl-sn-glycerol 3-phosphate) in guinea pig peritoneal polymorphonuclear leukocytes


Quantitative analysis of platelet-activating factor in rat brain


Residue analysis of oleic acid in cucumber fruits after spraying sodium oleate solution

Residue of cis-9-octadecenoic acid (ODA) was studied by gas chromatographic analysis of cucumber fruit sprayed with sodium oleate (20% liquid formulation), an aphicide under development at present in our laboratory. Endogenous ODA was 0.33 and 0.37ppm in two cultivars of cucumber fruit. Residue levels on the first day after last spraying were 1.81 and 3.57ppm, and on the third day they lowered to 0.88 and 1.01ppm. Recovery of ODA from untreated cucumber fruit to which 2.5ppm ODA had been added was in a range of 102.0 to 108.4%.

アブラムシ防除剤として開発中のオレイン酸ナトリウム液剤 (主成分としてシス-9-オクタデセン酸ナトリウムを20%含む) のキュウリ散布後のシス-9-オクタデセン酸 (ODA) の残留量を調べるため, ガスクロマトグラフによる分析を行なった. 二品種のキュウリ中の内因性ODA量は, それぞれ0.33, 0.37ppmであった. 二品種のキュウリ中のODA残留量は, 最終散布後1日目はそれぞれ1.81, 3.57ppmであり, 3日目においては, それぞれ0.88, 1.01ppmへと減少した. 未処理のキュウリにODAを2.5ppm添加し回収率試験を行なったところ, 回収率は102.0∼108.4%の範囲にあった.


Age-related decrease of the platelet-activating factor (PAF) content in rat brain was shown by a convenient method consisting of solid extraction of lipids with a Sep-Pak C-18 cartridge, lipid separation by HPLC and bioassay on rabbit platelets. This method was sufficiently sensitive to allow measurement of PAF in a single brain, and the recovery of PAF was quite high throughout the procedure.


Antagonism of platelet-activating factor in isolated rat colon: possible mechanism

The contractions of three different regions of rat colon in response to platelet-activating factor (PAF) were compared. The ascending colon was found to be the most responsive. The slow contraction of the ascending colon induced by PAF was dependent on external Ca2+. CV-3988, a structural analog of PAF, slowly induced irreversible inhibition of PAF-induced contraction, whereas FR-900452, which is structurally unrelated to PAF, caused rapid reversible inhibition of PAF-induced contraction. No inhibitory effects of CV-3988 were observed when the strip was washed with Tyrode's solution containing 1% bovine serum albumin (BSA). The results suggest that PAF and CV-3988 penetrate slowly into the outer half of the lipid bilayer of plasma membranes of cells in isolated rat colon, and then rapidly diffuse laterally to associate firmly with specific binding sites.


Lysophosphatidic acids induce contraction of rat isolated colon by two different mechanisms

Lysophosphatidic acids (1-linoleoyl-, 1-linolenoyl, 1-arachidonoyl- and 1-O-hexadecyl-sn-glycero-3-phosphate) induced rapid contraction of rat isolated colon which was dependent on external Ca2+, 1-linolenoyl-lysophosphatidic acid having the greatest effect. The contraction induced by 1-linolenoyl-lysophosphatidic acid was reduced considerably by nifedipine and verapamil, but not by atropine or indomethacin. Phosphatidic acids with two short-chain acyl groups induced a small, atropine-sensitive contraction at 100 microM, but phosphatidic acids with two long-chain acyl groups were inactive. These results suggest that unlike phosphatidic acids, lysophosphatidic acids act directly on extracellular sites of the plasma membrane of smooth muscle cells in rat colon, mainly through activation of voltage-sensitive Ca2+ channels.

The Journal of Pharmacy and Pharmacology


Identification of sn-2-ω-hydroxycarboxylate-containing phospholipids in a lipid extract from bovine brain

Phospholipids having both a long-chain acyl (palmitoyl or stearoyl) and a short-chain hydroxycarboxylyl (C3-C9) residue were identified by GC-MS in a fraction with PAF-like activity from a bovine brain lipid extract. The hydroxyl group in the hydroxycarboxylate residue was determined to be at the omega-position by comparison of the mass spectra of the tert-butyl-dimethylsilyl derivatives of these compounds with those of synthetic hydroxybutyrate-containing phosphatidylcholines. The co-existence of short-chain hydroxycarboxylate-, monocarboxylate- and dicarboxylate-containing phospholipids in the bovine brain lipid extract suggested that these compounds were formed by peroxidation of membrane phospholipids, especially phosphatidylcholines.

Biochemical and Biophysical Research Communications


Translocation of exogenous platelet-activating factor and its lyso-compound through plasma membranes is a rate-limiting step for their metabolic conversions into alkylacylglycerophosphocholines in rabbit platelets and guinea-pig leukocytes

Platelets and leukocytes are known to degrade platelet-activating factor (PAF), a potential mediator of inflammation, to its lyso-derivative (lyso-PAF) and then convert this to 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholines. However, little is known about the mechanism of internalization of PAF and lyso-PAF, which is a prerequisite for their metabolism within the cells. In this work, the internalization of PAF and lyso-PAF by rabbit platelet and guinea-pig leukocyte plasma-membranes were examined by the washing method with bovine serum albumin. The rates of translocation of PAF and lyso-PAF across guinea-pig plasma membranes were significantly higher than those across rabbit platelets. In these cells, the translocation of PAF was found to be accelerated indirectly by activation of PAF receptors by a small portion of added PAF. Results suggest that a temperature-dependent diffusion process is involved in the internalization of these phospholipids. In both rabbit platelets and guinea-pig leukocytes, the translocation of PAF and lyso-PAF through the plasma membranes was shown to be rate-limiting for the metabolic conversion of these compounds to 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine.

Biochimica et Biophysica Acta (BBA) - Bioenergetics


Calcium movements in rabbit platelets induced by platelet-activating factor and its regulation by an antagonist of platelet-activating factor,


Vitamin E. deficiency increases the synthesis of platelet-activating factor (PAF) in rat polymorphonuclear leucocytes

Vitamin E deficiency was found to stimulate FMLP (N-formyl-L-methionyl-L-leucyl-L-phenylalanine)-induced biosynthesis of PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in polymorphonuclear leucocytes (PMN) from rat peritoneum. In three separate experiments each, the amounts of PAF synthesized during 6 min and 12 min incubation of PMN cells from control, vitamin E-supplemented, and vitamin E-deficient rats were 129-240, 131-227 and 248-354 pmol/10(6) cells, respectively. The activity of the acetyl-transferase, which transfers the acetyl moiety of [3H]acetyl-CoA to 2-lysoPAF (1-O-alkyl-sn-glycero-3-phosphocholine) to form [3H]PAF, was higher in PMN homogenates from vitamin E-deficient rats (2.28 +/- 0.07 nmol/min/mg protein) than in those from E-supplemented rats (1.06 +/- 0.10 nmol/min/mg protein). However, there was no difference between the two groups in the activity of acetylhydrolase (4.26 +/- 0.71 and 4.26 +/- 0.06 nmol/min/mg protein, respectively), measured as degradation of [3H]PAF to [3H]lysoPAF. In vitro addition of alpha-tocopherol did not inhibit the increased activity of acetyl-transferase in vitamin E-deficient rats, indicating that the enzyme in vitamin E-supplemented rats was not directly inhibited by alpha-tocopherol. The acetyl-transferases of the two groups showed similar Km values for acetyl-CoA, but different Vmax values (225 microM and 6.4 nmol/min/mg protein in vitamin E-deficient rats, and 216 microM and 3.6 nmol/min/mg protein in vitamin E-supplemented rats), suggesting that the enzyme was not activated but increased in amount in vitamin E deficiency.


Novel molecular analogues of phosphatidylcholines in a lipid extract from bovine brain: 1-long-chain acyl-2-short-chain acyl-sn-glycero-3- phosphocholines


Novel phospholipids with aliphatic dicarboxylic acid residues in a lipid extract from bovine brain

A vasodepressor phospholipid fraction purified from a lipid extract of bovine brain was found to contain novel phospholipids with both a long-chain acyl group and an aliphatic dicarboxylic acid residue. This was shown by analyzing the fraction as tert-butyldimethylsilyl derivatives of glyceride by capillary GC-MS after hydrolysis with phospholipase C. Six molecular species with a palmitoyl group and an aliphatic dicarboxylate (chain length C4-C9), and two species with both a stearoyl group and a succinate or glutarate residue were detected.


Study of platelet activating factor and its antagonists on rat colon strip with a new method avoiding tachyphylaxis

Platelet activating factor induced slow, but sustained contraction of isolated rat colon in a dose-dependent manner. The contraction persisted even when the strips were washed several times with Tyrode solution. However, the addition of high concentrations of bovine serum albumin to the washing solution led to the rapid relaxation of the strips to the basal level, possibly by removing platelet activating factor tightly bound to rat colon. These strips then responded normally to a second addition of platelet activating factor. Neither the cholinergic nervous system nor release of histamine, serotonin, prostaglandins or leukotriene D4 were significantly involved in the contraction induced by platelet activating factor. FR-900452 and CV-3988, recently found to be antagonists of platelet activating factor, selectively blocked the contraction of rat colon induced by the active phospholipid, indicating that it induced contraction by a direct stimulatory effect on the smooth muscle of rat colon in a receptor-mediated manner.


The effect of a-tocopherol on site-specific lipid peroxidation induced by iron in charged micelles

alpha-Tocopherol inhibited H2O2-Fe2+-induced lipid peroxidation of linoleic acid (LA) by scavenging OH radicals in tetradecyltrimethylammonium bromide (TTAB) micelles. The inhibiting ability of alpha-tocopherol was much greater than that of OH-radical scavengers mannitol and t-butanol. In contrast, alpha-tocopherol enhanced linoleic acid hydroperoxide (LOOH)-Fe2+-induced lipid peroxidation through regeneration of Fe2+ in sodium dodecyl sulfate (SDS) micelles containing LA. alpha-Tocopherol was oxidized by Fenton's reagent (FeSO4 + H2O2) at a higher rate in SDS micelles than in TTAB micelles. The likely oxidants were OH radicals in the former and Fe3+ in the latter. Both reagents formed in the Fenton reaction. Ferrous ion catalyzed in a dose-dependent manner the decomposition of LOOH and conjugated diene compounds in SDS but not in TTAB micelles. alpha-Tocopherol and Fe3+ individually had no effect on the decomposition of LOOH, but together were quite effective. The rate of the decomposition was a function of the concentration of alpha-tocopherol. The mechanism of "site-specific" antioxidant action of alpha-tocopherol in charged micelles is discussed.


Binding and internalization of platelet-activating factor 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine in washed rabbit platelets

The binding profile of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC, platelet-activating factor) to washed rabbit platelets was investigated through the use of structural analogs of AGEPC, e.g. U66985, which specifically suppressed AGEPC biological activities on rabbit platelets. This interaction of AGEPC with platelets could be divided into three different components termed A, B, and C. Component A was considered as one of high affinity (Kd = 0.5 X 10(-9) M) and with a low capacity (about 400 sites/platelet). The binding of AGEPC to component A was reversible and was blocked by the inhibitory analogs of AGEPC. This was considered to be the AGEPC receptor site(s). Component B was irreversible in nature and was presumed to be associated with internalization of AGEPC. The latter process was sensitive to the structural inhibitors. Component C was not affected by the inhibitors and probably represented a nonspecific binding to the lipid layer of the membrane. The binding profile of 1-O-alkyl-2-(lyso)-sn-glycero-3-phosphocholine, a biologically inactive and noninhibitory analog of AGEPC, was observed to consist of a single component and was (also) unaffected by the inhibitors. Internalization of AGEPC into rabbit platelets was further examined by the bovine serum albumin extraction method, which was originally developed by Mohandas et al. (Mohandas, N., Wyatt, J., Mel, S. F., Rossi, M. E., and Shohet, S. B. (1982) J. Biol. Chem. 257, 6537-6543). AGEPC was instantly taken up by the cell and internalization into its membrane, where it remained and was not released into cytosol. The internalization of AGEPC was suppressed by pretreating the cells with AGEPC analogs. In platelets desensitized to AGEPC, no down-regulation of the receptor site(s) was observed. The internalization of AGEPC in the desensitized cells was clearly enhanced and this was obvious even in the presence of the AGEPC inhibitor(s). Even in the presence of the inhibitors, effective internalization of AGEPC was also evident in thrombin-treated cells. These results suggested that the internalization of AGEPC was irreversibly enhanced in the platelets which were activated by AGEPC itself as well as by thrombin.


Evidence for existence of various homologues and analogues of platelet activating factor in a lipid extract of bovine brain

Vasodepressor phospholipid with platelet-aggregating activity was highly purified from a lipid extract of bovine brain and subjected to field desorption-mass spectrometry. It was further analyzed by gas-liquid chromatography-mass spectrometry after hydrolysis with phospholipase C and conversion to tert-butyldimethylsilyl derivatives. Results indicated the presence of four species of platelet activating factor (1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine, PAF) and ten acyl analogues of PAF. The acyl analogues of PAF included species having an sn-2-propionyl or sn-2-butyryl group, which have not been previously detected in natural sources. The total amount of acyl analogues of PAF was much higher than that of PAF.

Biochemical and Biophysical Research Communications


Platelet aggregation induced by ether-linked phospholipids. 1. Inhibitory actions of bovine serum albumin and structural analogues of platelet activating factor

Ether-linked lysophosphatidic acid was able to induce aggregation of platelets from various animals. Feline platelets were the most sensitive followed in decreasing order by human, bovine and rabbit platelets. ONO-6240 and CV-3988, which are antagonists of platelet activating factor, did not inhibit aggregation of feline platelets induced by ether-linked lysophosphatidic acid, indicating that the aggregation induced by the lysophosphatidic acid did not involve receptors for platelet activating factor. An addition of bovine serum albumin caused dose-dependent inhibitions on the aggregation of platelets by both ether-linked lysophosphatidic acid and platelet activating factor. Moreover, 0.5% bovine serum albumin reversed platelet aggregation by the active phospholipid before this aggregation reached a maximum. These results suggest that the lysophosphatidic acid exerts its stimulatory action extracellularly, probably by interacting with specific sites on the surface of platelet.


Platelet aggregation induced by ether-linked phospholipids. 2. Mechanism of desensitization of rabbit platelets by platelet activating factor and reversibility of inhibitory actions of its antagonists

Rabbit platelets pretreated with CV-3988 had reduced responsiveness to platelet activating factor(PAF) even after the platelets had been washed with a buffer. This was due to its tight binding to receptor sites for PAF, but not conformational changes of the receptor, because the platelets that were pretreated with CV-3988 and then washed with buffer containing 0.5% bovine serum albumin showed full responsiveness to PAF. In contrast, the reduced responsiveness of PAF-pretreated rabbit platelets was not reversed by washing with buffer containing 0.5% BSA. The PAF-pretreated platelets showed significantly lower responsiveness to 1-O-hexadecyl-2-O-monomethylaminocarbonyl-sn-glycero-3-phosphochol ine (MC-PAF) than to PAF, although MC-PAF had the same potency as PAF on inducing aggregation of normal rabbit platelets by interacting with receptor sites for PAF. These results suggest that the desensitization is accompanied by a decreased affinity of receptor sites for PAF or reduced efficiency in coupling of activated receptor to regulatory protein in plasma membranes rather than loss of receptor sites.


A platelet-aggregating and hypotensive phospholipid isolated from bovine brain

A phospholipid that differs from known active lipids and causes potent platelet aggregation and weak hypotension has been isolated from bovine brain. Its platelet aggregating effect on heparinized platelet-rich plasma from rabbits, was at a threshold concentration of about 0.2 nmol ml-1 as phosphorus. The effect was inhibited by CV-3988. The phospholipid was converted by diazomethane treatment to another active lipid that caused short-term hypotension, but not platelet aggregation, rather it inhibited the aggregation of rabbit heparinized platelets induced by platelet-activating factor.

The Journal of Pharmacy and Pharmacology


Alkylacetylglycerophosphocholine effects on the metabolism of phospholipids in rabbit platelets: effects of extracellular Ca2+ and prostacyclin

Alkylacetylglycerophosphocholine (AGEPC) stimulation of 32P-labeled lysophosphatidic acid formation in washed rabbit platelets was dependent on extracellular Ca2+. Its accumulation was slower and required a higher concentration of AGEPC in comparison to the degradation of inositol phospholipids and production of phosphatidic acid induced by the same agonist. These results suggest that the formation of lysophosphatidic acid is not directly related to the primary activation of rabbit platelets by AGEPC. AGEPC elicited a preferential degradation of inositol phospholipids in the following order: phosphatidylinositol 4,5-bisphosphate greater than phosphatidylinositol 4-phosphate greater than phosphatidylinositol. The degradation of inositol phospholipids and subsequent production of phosphatidic acid were affected by pretreatment of platelets with prostacyclin or ethylene glycol bis (beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). Synergistic inhibitions of these metabolic changes were observed in the platelets pretreated with both prostacyclin and EGTA. These results were compared with effects of prostacyclin and EGTA on serotonin release induced by AGEPC, and the possible roles of metabolic changes in phospholipids induced by AGEPC are discussed with respect to the mechanism of platelet activation.


Gas chromatographic/mass spectrometric analyses of ether-linked lysophosphatidic acid and its analogues

Biomedical & Environmental Mass Spectrometry


Involvement of lysophospholipase D in the production of lysophosphatidic acid in rat plasma.

Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism


Fluorescent pigments by covalent binding of lipid peroxidation by-products to protein and amino acids

The fluorescent products formed on reaction of 12-oxo-cis-9-octadecenoic acid (12-keto-oleic acid) with about 20 different amino acids, polylysine and bovine serum albumin (BSA) were studied. Besides glycine, only the basic amino acids histidine, lysine and arginine gave products with strong fluorescence. N-Acetylation of amino acids greatly reduced the fluorescence of their reaction products. The formation of fluorescent products was inhibited strongly by SH-amino acids such as N-acetyl-cysteine and glutathione. Polyacrylamide gel electrophoresis showed that BSA treated with 12-keto-oleic acid was more acidic than untreated or ricinoleic acid-treated BSA, indicating that basic amino acid residues in BSA were modified by reaction with the keto fatty acid. None of the structural analogs of 12-keto-oleic acid tested--12-oxo-trans-10-octadecenoic acid, 12-oxo-octadecanoic acid, 12-hydroxy-cis-9-octadecenoic acid (ricinoleic acid), cis-9-octadecenoic acid (oleic acid) and linoleic acid--reacted with glycine to give a fluorescent product. The fluorescent products formed on reaction of 12-keto-oleic acid methyl ester with benzyl amine and glycine methyl ester were shown to be 8-(N-substituted-4,5-dihydro-4-oxo-5-hexyl-5-hydroxy-2-pyrrolyl) octanoic acid methyl esters. The fluorescence properties of these compounds were attributed to the chromophobic system NC = CC = O which contains 6 pi electrons. This investigation contributes to insight of the mechanism of formation of fluorescent pigments, probably by a similar reaction of other compounds of the beta, gamma-unsaturated carbonyl type.


A novel approach to structure proof of glyceryl ether-containing glycerophospholipids. Base-catalyzed methanolysis of platelet-activating factor (AGEPC) at 60 degrees C

A novel reaction was explored in which synthetic platelet-activating factor, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC), upon treatment with 1 N NaOH in methanol at 60 degrees C for 20 min, sequentially released the acetyl group, then the choline moiety with concomitant formation of the monomethyl ester of 1-O-alkyl-glycero-phosphoric acid. A mechanism is proposed in which a transient cyclic phosphate intermediate is formed and then attacked by a CH3O moiety to yield a mixture of the sn-2 and sn-3 methyl esters. Proof of structure of the monomethyl ester derivative was achieved through the use of thin-layer chromatography, aluminum oxide chromatography, and examination of the trimethylsilyl derivative of the monomethyl ester by gas-liquid chromatography-mass spectrometry. Replacement of the acyl group on the 2 position with an ethyl or methyl residue completely prevented any attack by 1 N NaOH in methanol at 60 degrees C. Sphingomyelin was not attacked and only acetate removal was noted with 1-O-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamine under similar conditions. The significance of these findings as they relate to the influence of substituents on the chemical and biological reactivity of AGEPC is discussed.


Structural analogs of alkylacetylglycerophosphocholine inhibitory behavior on platelet activation

Several analogs of alkylacetylglycerophosphocholine (AGEPC; platelet-activating factor) were investigated as potential selective inhibitors of AGEPC-induced activation of washed rabbit platelets. Two particular compounds, CV-3988 (rac-3-(N-n-octadecylcarbamoyloxy)-2-methoxypropyl-2-thiazolioethyl++ + phosphate) and U66985 (1-O-octadecyl-2-acetyl-sn-glycero-3-phosphoric acid-6'-trimethylammonium-hexyl ester) emerged as particularly active and effective inhibitors. Aggregation and secretion profiles, as well as the degradation of inositol phospholipids and production of phosphatidic acid, were used as monitors of their inhibitory capabilities. U66985 was the most effective inhibitor, giving an IC50 value of 4.1 +/- 1.5 X 10(-8) M against a challenge of 1 X 10(-10) M AGEPC in the secretion assay. Phospholipid turnover was blocked completely at this inhibitor concentration. On the other hand, while CV-3988 was an effective inhibitor, a higher concentration was required and a more restricted range of activity was noted with an IC50 value of 5.9 +/- 1.3 X 10(-7) M against a challenge of 1 X 10(-10) M AGEPC in the secretion assay. While CV-3988 did indeed completely block the turnover of inositol phospholipids and phosphatidic acid formation, these effects were noted at a higher concentration than with U66985. On the basis of data obtained in desensitization experiments with AGEPC and U66985, it appears that each inhibitor occupies the same receptor site as the agonist, AGEPC. These results illustrate the usefulness of these AGEPC analogs in exploring the biochemical characteristics of the interaction of AGEPC with a cell.


Gas chromatographic-mass spectrometric analysis of biologically active phospholipids having an sn-2-acetyl group.


Identification of steroidal compounds as acetates in a lysate of "Depressor-I," an antihypertensive phospholipid occurring in acetone-soluble fraction of bovine brain

The alkaline hydrolysate of "Depressor-I", an antihypertensive phospholipid occurring in the acetone-soluble fraction of bovine brain, was [3H] acetylated and applied to an alumina column. Various [3H] acetylated compounds were eluted. Among the major fractions, the eluates with hexane and hexane-benzene (9 : 1, 7 : 3 and 5 : 5, v/v) mixtures contained labile compounds which decomposed on standing for one month. Gas chromatography-mass spectrometry was carried out with the corresponding sample obtained in a "cold"experiment, and diosgenin acetate, a sapogenin, was detected besides cholesteryl acetate. A new class of phospholipid containing steroidal components is proposed.


Isolation of diosgenin[(25R)-spirost-5-en-3 beta-ol] from a lysate of a new hypotensive phospholipid occurring in bovine brain

Diosgenin[(25R)-spirost-5-en-3 beta-ol] and cholesterol were detected as trimethylsilyl derivatives by gas chromatography-mass spectrometry in an alkaline lysate of a new hypotensive phospholipid from the lipid fraction of bovine brain. These steroids seemed to be present in the hypotensive phospholipid fraction in some bound forms.


Stimulation of (Ca2++Mg2+)-ATPase in human erythrocytes membranes by synthetic lysophosphatidic acids and lysophosphatidylcholines. Effects of chain length and degree of unsaturation

Synthetic lysophosphatidic acids and lysophosphatidylcholines were examined for their effects on the (Ca2+ + Mg2+)-ATPase of human erythrocyte membranes. Addition of these compounds to erythrocyte ghosts caused significant changes in ATPase activity. The degree of unsaturation and the length of the sn-1 long chain hydrocarbon moiety were both contributing factors. All lysophosphatidic acids tested stimulated (Ca2+ + Mg2+)-ATPase activity. Of the species having a saturated acyl group, the most active was the myristoyl derivative. Linoleoyllysophosphatidic acid was the most potent of the unsaturated species. Saturated lysophosphatidylcholines with a short chain fatty acyl group (C10 to C14) exhibited only a moderate stimulatory activity, whereas the longer chain homologues, i.e., C16 and C18 were inhibitory at high concentrations. On the other hand, unsaturated lysophosphatidylcholines had stimulatory activities comparable to the unsaturated lysophosphatidic acids. These results suggest that the acidic moiety of lysophosphatidic acid is not an important structural determinant for expressing ATPase stimulatory activity in ghosts. Rather the nature of the hydrocarbon chain as well as the lyso structure of these compounds appear most critical under these conditions. The stimulatory effects of lysophosphatidic acids or lysophosphatidylcholines were additive to that induced with calmodulin, suggesting that these lysophospholipids affect the (Ca2+ + Mg2+)-ATPase by a mechanism which is different from that seen with calmodulin.

Biochimica et Biophysica Acta (BBA) - Biomembranes


Effects of lysophosphatidic acids and their structural analogs on arterial blood pressure of cats

On intravenous injection into cats, lysophosphatidic acid elicited a biphasic change in arterial blood pressure: sharp hypotension followed immediately by hypertension. Respiration was greatly disturbed immediately after injection of lysophosphatidic acid, and remained stimulated for a long period, and the heart rate increased during the period of hypertension. Unsaturated lysophosphatidic acids were more potent in evoking hypertension than saturated ones. The hypotensive activity of 1-palmitoyl-2-0-acetyl-sn-glycero-3-phosphate was about half that of 1-palmitoyl-2-lyso-sn-glycero-3-phosphate (lysophosphatidic acid). Elongation of the acyl chain at the sn-2-position of the glycerol moiety resulted in progressive reduction in the hypotensive activity. Chemically modified analogs with a head group, such as phosphoryl-methanol, ethanol, propanol and choline, had little or no activity. However, sn-2-acetyl-analogs of lyso-phosphatidyl cholines had hypotensive activity. 1-0-Hexadecyl-2-lyso-sn-glycero-3-phosphate, an alkyl-lyso-phosphatidic acid, had much stronger hypotensive activity than the corresponding acyl-lysophosphatidic acid, like its sn-2-0-acetyl analog. These structure-activity relationships of lysophosphatidic acids indicate the existence of their specific binding sites on cardiovascular cells.


Comparison of antispasmodic effect of synthetic lysolecithins with various fatty acid moieties on guinea pig ileum

Non-competitive antispasmodic effects of 8 kinds of synthetic L-alpha-lysolecithins (1-O-acyl-2-lyso-sn-glycero-3-phosphocholine, LPC) with various fatty acid moieties were examined upon the spasmodic actions of histamine and acetylcholine on guinea pig ileum. Five-min pretreatment of the gut with the synthetic LPC was required to be effective. With increase of the concentration of a synthetic LPC for pretreatment of the ileum, the slope of the dose-response curve of histamine or acetylcholine added cumulatively became more gentle, the maximal contraction was suppressed apparently, and which were associated with the shift of the curve to the higher concentration of the stimulants. Of LPCs with saturated fatty acid palmitoyl-LPC showed the strongest effect, followed by myristoyl-, stearoyl-, lauroyl- and decanoyl-LPCs in order. Incorporation of cis-double bond into the C18 fatty acid chain of LPC resulted in slight decrease of the antispasmodic effect. The relaxating effect of LPCs on perfused rabbit ear vessel preparations was similar in order.


Analyses of lysophosphatidic acids by gas chromatography mass spectrometry without hydrolytic pretreatment

Lysophosphatidic acids having different fatty acyl moieties were directly analysed by gas chromatography/mass spectrometry after silylation. Trimethylsilyl derivatives of various LPAs escaped thermal degradation, and electron impact and chemical ionization of these derivatives yielded many ions which were useful for the structural determination. It was found by this new method that five species of lysophosphatidic acids were formed in rat plasma during incubation at 37 degrees C.


Contractile action of 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine on strips of isolated rat intestine

The contractile effects of AGEPC were examined on various regions of rat isolated intestine. The duodenum, jejunum and ileum showed only the tonic component of contraction to AGEPC at the low dose (less than 10(-9) M) but at the high dose (10(-7) M) biphasic contractions were induced, consisting of a phasic followed by a tonic component. In the colon, however, the AGEPC-induced maximum contraction was comparable in magnitude to that produced by acetylcholine; also the contraction profile was different from that elicited from the other regions of the intestine. Low doses of AGEPC caused a slow, sustained contraction and at high doses phasic and tonic components were not dissociated.

The Journal of Pharmacy and Pharmacology


Mass spectrometric analyses of biologically active choline phospholipids and their lysoderivatives

The electron impact and chemical ionization mass spectra of 1-O-hexadecyl- and 1-palmitoyl-2-O-acetyl-sn-glycero-3-phosphocholines and their lyso derivatives were measured by insertion of the compounds into a direct inlet system. The lysophospholipids were decomposed by heating at over 300℃ into multiple compounds that volatilized together and gave several characteristic ion peaks when subjected to electron impact or interaction with an ion plasma of reactant gas. The mass spectral data indicated that the major pyrolysis products of these lysophospholipids were produced by elimination of methanol or N, N-dimethylethanolamine. When sn-2-acetyl phospholipids were introduced on the direct insertion probe and heated, several pyrolysis products volatilized together at above 350℃. The results suggested the major pyrolysis mechanism was loss of the phosphorylcholine moiety, together with some deacetylation and subsequent elimination of methanol and N, N-dimethylethanolamine from the sn-2-acetyl phospholipids.


Biphasic action of platelet-activating factor on isolated guinea-pig ileum

1-0-Hexadecyl-2-0-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) at 10(-10)-10(-9) M induced slow contraction of isolated guinea-pig ileal muscles and the contraction persisted for a long time. At a higher concentration of 10(-7) M, this phospholipid induced more rapid, but not greater, contraction. At higher concentrations (10(-6)-10(-5) M), this phospholipid induced a biphasic response: rapid contraction followed by relaxation. At high concentrations, this compound inhibited acetyl-choline-induced contractions. The stimulatory effect of this phospholipid was ca. 300 times that of 1-palmitoyl-2-0-acetyl-sn-glycero-3-phosphocholine, while its inhibitory potency on induced contraction was similar to those of 1-palmitoyl-2-0-acetyl-sn-glycero-3-phosphocholine and its lyso derivative. It was suggested that the differences in effects on contraction of different concentrations of 1-0-hexadecyl- and 1-palmitoyl-2-0-acetyl-sn-glycero-3-phosphocholine were due to the effects of these compounds on the ileum: a strong stimulatory effect and a moderate inhibitory effect on contraction.


Contractile actions of lysophosphatidic acids with a chemically-defined fatty acyl group on longitudinal muscle from guinea-pig ileum.

Various lysophosphatidic acids caused phasic contractions of longitudinal muscle from guinea-pig ileum, followed by rhythmical contractions of lower magnitude. Polyunsaturated lysophosphatidic acids were more potent than saturated lysophosphatidic acids. Among the lysophosphatidic acids tested, linolenoyl-lysophosphatidic acid was the most active, but its activity was about 3-5 times less than that of prostaglandin F2 alpha. Contractile responses to various lysophosphatidic acids, were partially suppressed by tetrodotoxin, atropine and chlorpheniramine. In addition, lysophosphatidic acid-induced contractions were considerably reduced by pretreatment of the muscle with indomethacin.


Antioxidant activities of tocopherols on Fe2+-ascorbate-induced lipid peroxidation in lecithin liposomes

The antioxidant activities of 4 tocopherols, tocol, and a water-soluble model analog of alpha-tocopherol were compared. Egg lecithin liposomes were used and oxidation was catalyzed by Fe2+-ascorbate. The activities decreased in the order alpha greater than beta greater than gamma greater than delta-tocopherol greater than tocol, in agreement with their potencies in vivo. The water-soluble analog was the least effective. Activity depended on the molar ratio of antioxidant to unsaturated lipid, with one molecule each of the alpha-, beta-, gamma-, delta-tocopherol and tocol capable of protecting, respectively, 220, 120, 100, 30 and 20 molecules of polyunsaturated fatty acid. The mechanism of possible antioxidant effect of the compounds used is discussed.


Mass Spectrometric analyses of lysophosphatidic acids and their dimethyl ester


Cardiovascular effects of lysophosphatidic acid and its structural analogs in rats

Injection of lysophosphatidic acid into anesthetized rats induced immediate hypertension, the effect depending on the structure of the sn-1-acyl moiety of the molecule. A hydroxyl group at the sn-2-position was not necessary, but a wedgeshaped structure was suitable for hypertensive activity. Most lysophospholipids with a chemical group attached to the phosphate portion had only weak hypotensive effects, but the sn-2-acetylated analogs of these depressor lysophospholipids elicited a hypotension at much lower doses. The durations of the hypotensions evoked by the sn-2-acetylated choline phospholipids were longer than those produced by 1-palmitoyl-2-acetoyl-sn-glycerol-3-phosphorylmethanol, ethanol and propanol.

The Journal of Pharmacology and Experimental Therapeutics


Lysophosphatidic acid-induced aggregation of human and feline platelets: Structure-activity relationship

Biochemical and Biophysical Research Communications


Antioxidative. effect of a-tocopherol incorporation into. lecithin liposomes on ascorbic acid-. Fe2+-induced lipid peroxidation


市販リン脂質中のグリセリン酸の分解

市販リン脂質中のグリセリン酸の分解

Journal of the Pharmaceutical Society of Japan


Stimulatory effect of lysophosphatidic acids on uterine smooth muscles of non-pregant rats

Lysophosphatidic acids stimulated isolated uterine smooth muscle dose-dependently. The contractions were not reduced by pretreatment with atropine or an anti-5-hydroxytryptaminic agent. The potency depended on the nature of the acyl chain in the molecule. Of the compounds with a saturated fatty acyl group tested, the most effective were myristoyl- and lauroyl-lysophosphatidic acid. In a series of unsaturated lysophosphatidic acids, the potency increased with the number of cis double bonds in the acyl chain, and linolenoyl-lysophosphatidic acid was the most active. When injected intravenously, these compounds induced an immediate rise in blood pressure and intrauterine pressure, like prostaglandin F2 alpha: The order of potency of their effects on the intact uterus was consistent with that of their effects on isolated uterine smooth muscle, but not with that of their hypertensive effects in rats.

Archives internationales de Pharmacodymie et de Therapie


Occurrence of short-duration hypotensive phospholipid from dog peritoneal dialysate

From the total lipid fraction of dog peritoneal dialysate after freeze drying and extraction, a hypotensive phospholipid was isolated through silicic acid, cellulose, and Sephadex LH-20 column chromatography in a pure grade; it showed a single spot on TLC. The purified hypotensive factor, designated as Peritoneal Dialysate Depressor-I, elicited potent depressor responses in anesthetized rats, and its threshold dose was approximately 35 microgram/kg. The material resisted proteases and 15-hydroxyprostanoate oxidoreductase. In mobility on TLC, the hypotensive factor was distinuishable from water-soluble hypotensive substances and also from depressor lipids. Judging from its behavior on TLC and column chromatography during the purification procedure, the hypotensive factor seems to be a choline-containing phospholipid and shows the general characteristics of hysolecithin, except for its potent hypotensive activity, and 2',7'-dichlorofluorescein on TLC. The molar ratio of phosphorusurated ones such as stearic and palmitic acids.


Action of depressor-I, a hypotensive phospholipid from bovine brain, on systemic and arterial blood pressures of various species

Effects of "Depressor-I" (D-I), a new hypotensive phospholipid obtained from bovine brain lipid fraction, on systemic arterial blood pressure were investigated. The hypotensive activity of D-I in urethane anaesthetized rats was dose dependent and tachyphylaxis and/or sensitization were not observed. Increments of the respiration and the heart rate were observed with sharp falls in blood pressures following intravenous administration of D-I, in simultaneous recordings in anaesthetized rats. D-I elicited hypotension in all species of animals examined, and the sensitivities to D-I were much the same, however, there were two types in patterns of duration on responses and the durations were also dose dependent. D-I exhibited depressor-responses even in conscious rats, though responses were much smaller compared with those seen in anaesthetized rats. In a comparison of anaesthetic agents in rats, the highest hypotensive activity of D-I was observed with pentobarbital anaesthesia, a moderate response was seen with alpha-chloralose and the least response was seen with urethane. In spinal rats or those pretreated with reserpine or antagonists, such as atropine, diphenhydramine, propranolol and hexamethonium, D-I also elicited hypotension. These results suggest that "Depressor-I" does not elicit the depressor action via the stimulation of the central and the autonomic nervous systems but rather by a direct action on peripheral blood vessels.


A hypotensive phospholipid from dog peritoneal dialysate

The Journal of Pharmacy and Pharmacology


Effect of α-tocopherol incorporation on glucose permeability and phase transition of lecithin liposomes

Liposomes were prepared from dipalmitoyllecithin, dimyristoyllecithin, dioleoyllecithin, egg lecithin, and soybean lecithin, and the effects of incorporation of various quantities of alpha-tocopherol or its analogs on permeability of the liposomes to glucose were studied at various temperatures (4--40 degrees C). Results showed that increase in the quantity of alpha-tocopherol incorporated into dipalmitoyllecithin and dimyristoyllecithin liposomes lowered the transition temperature for marked release of glucose and also decreased the maximum rate of temperature-dependent permeability, alpha-Tocopherol also had similar but less marked effects on the permeability of dioleoyllecithin and egg lecithin liposomes, but little effect on those of soybean lecithin, which has a higher degree of unsaturation. In dipalmitoyllecithin liposomes phytol showed a similar effect of permeability to that of alpha-tocopherol, but phytanic acid caused a different pattern of temperature-dependent permeability. With analogs of alpha-tocopherol, the regulatory effect on permeability decreased with shortening and disappearance of the isoprenoid side chain. The significance of these observations is discussed in relation to the physiological functions of tocopherols in natural membranes.


Physico-chemical characteristics of D-I, a hypotensive factor occurred in acetone extract of the bovine brain


Effects of synthetic and natural lysophosphatidic acids on the arterial blood pressure of different animal species

Intravenous injection of lysophosphatidic acid was found to cause hypertension in rats and guinea pigs, but hypotension in cats and rabbits. The potencies of the pressor and depressor effects of synthetic lysophosphatidic acids in rats and cats depended on their chain length and the degree of unsaturation of their fatty acyl moieties.


Identification of vasopressor phospholipid in crude soybean lecithin

The vasopressor phospholipid in crude soybean lecithin was isolated by column chromatography on Sephadex LH-20. It represented 0.1% of crude soybean lecithin. The isolated phospholipid was identified to be lysophosphatidic acid by gas chromatography-mass spectrometry analysis of TMS-deacylated product and acetolysis product. Nuclear magnetic resonance analysis favored the 1-monoacyl isomer over the 2-isomer. By enzymic determination with L-3-glycerophosphate dehydrogenase, the isolated phospholipid was identified as 1-monoacyl-L-3-glycerophosphate. Gas chromatographic examination revealed that it was composed of a large percentage of unsaturated fatty acids, especially linoleic acid. The activity of isolated lysophosphatidic acid was slightly less than that of synthetic 1-linoleoyl-L-3-glycerophosphate.


ドデシル硫酸ナトリウムの長期毒性試験

Weanling male rats were bred with a drinking water containing sodium dodecyl sulfate (SDS) at concentrations of 0.25,0.05,or 0% for 5 months. Increase of organ weights was observed in the lung and kidney, though body weight was not affected at a higher concentration (0.25%). Histopathology of major organs indicated the characteristic symptom to be bronchopneumonia by the continued ingestion of a small amount of SDS. Activities of serum enzymes, such as GOT, GPT, alkaline phosphatase, and cholinesterase, were not affected at the dosages used. In rats taking 0.25% of SDS, triglyceride level increased in the liver but decreased in serum, while hepatic and serum levels of cholesterol, phospholipid, and free fatty acid were unchanged.

Japanese Journal of Toxicology and Environmental Health


Chemical and pharmacological properties of vasopressor phospholipid in crude soybean lecithin

Commercial soybean lecithin has a potent hypertensive effect on anesthetized rats. Crude soybean lecithin was separated into two fractions with respect to solubility in cold acetone. The acetone-insoluble fraction containing most pressor activity was separated into two further active fractions by column chromatography on silicic acid. The active fraction eluted with chloroform-methanol mixture (4:6) was further purified by gel filtration on Sephadex LH-20. The purified pressor substance had phosphorus and was labile to alkali. Its chromatographic and partition behaviors indicate that it may be a polar acidic phospholipid. The short-lasting pressor response produced by the active lipid was not reduced by mechanical injury to the central nervous system or treatment with an a-adrenergic blocking agent or a ganglion-blocking agent.


Isolation of an acute hypotensive substance from bovine brain lipid fraction


Glutathione peroxidase activity in tissues of vitamin E-deficient mice

Journal of Nutritional Science and Vitaminology


D-Alanine in germinating Pisum sativum seedlings


Complexes of starch and its related materials with organic compounds. Part X, X-ray diffraction of amylose-fatty acid complexes


ニワトリ卵白のリゾホスホリパーゼDによるリゾホスファチジン酸の産生

ニワトリ卵白のリゾホスホリパーゼDによるリゾホスファチジン酸の産生

Proceedings of the Japanese Conference on the Biochemistry of Lipids


喫煙による循環血液での酸化リン脂質の産生ー血管病変への関与の可能性ー

喫煙による循環血液での酸化リン脂質の産生ー血管病変への関与の可能性ー

平成17年度喫煙科学研究財団研究年報

平成17年度喫煙科学研究財団研究年報


喫煙による循環血液での酸化リン脂質の産生ー血管病変への関与の可能性ー

喫煙による循環血液での酸化リン脂質の産生ー血管病変への関与の可能性ー

平成16年度喫煙科学研究財団研究年報

平成16年度喫煙科学研究財団研究年報


ウサギ房水のリゾホスホリパーゼD活性によるリゾホスファチジルコリンの分解

ウサギ房水のリゾホスホリパーゼD活性によるリゾホスファチジルコリンの分解


リン脂質酸化二次作物による血管平滑筋細胞のアポトーシス誘導とビタミンEによる抑制作用


抗ガン作用の増強を志向したビタミンEコハク酸ベシクル

抗ガン作用の増強を志向したビタミンEコハク酸ベシクル


喫煙による循環血液での酸化リン脂質の産生ー血管病変への関与の可能性ー

喫煙による循環血液での酸化リン脂質の産生ー血管病変への関与の可能性ー

平成15年度喫煙科学研究財団研究年報

平成15年度喫煙科学研究財団研究年報


Proinflammatory platelet activating factor-like phospholipids and their pathophysiological significance


正常および異常妊娠時のヒト血清リゾホスホリパーゼD活性

正常および異常妊娠時のヒト血清リゾホスホリパーゼD活性


生理活性リン脂質LPAを産生するヒト血漿リゾホスホリパーゼDは多機能性ヌクレオチドホスジエステラーゼautotaxinである


モルモット腹腔洗浄液のリゾホスホリパーゼD活性

モルモット腹腔洗浄液のリゾホスホリパーゼD活性


Enhancement by α-tocopherol hemisuccinate of nitric oxide production induced by lipopolysaccharide and interferon-γ through up-regulation of protein kinase C in rat vascular smooth muscle cells

Proc.Of the second China-Japan international Conference on Vitamins


Acyl and alkyl ether-linked lysophosphatidic acids in the nervous system

Annals of the New York Academy of Sciences


Lysophosphatidic Acids Produced by Lysophospholipase D in Mammalian Serum and Body Fluid


血清リゾホスホリパーゼDによるリゾホスファチジン酸産生の病態下での変動

血清リゾホスホリパーゼDによるリゾホスファチジン酸産生の病態下での変動


高度不飽和脂肪酸を有するリゾホスファチジン酸:血漿中での生成と血小板凝集活性

高度不飽和脂肪酸を有するリゾホスファチジン酸:血漿中での生成と血小板凝集活性


リポキシゲナーゼによるホスファチジルコリンの酸化分解

リポキシゲナーゼによるホスファチジルコリンの酸化分解


スーパーオキシド依存症の脂質過酸化を誘導する発癌性鉄錯体Fe-NTA(ニトリロ三酢酸)の特性


リボソーム膜におけるα-トコフェロールの一重項酸素脱活作用:反応速度定数の測定

リボソーム膜におけるα-トコフェロールの一重項酸素脱活作用:反応速度定数の測定


妊婦血清リボホスホリパーゼD活性

妊婦血清リボホスホリパーゼD活性


α-トコフェロールの立体異性体の分離および微量定量

α-トコフェロールの立体異性体の分離および微量定量


Fe2+/アスコルビン酸系ーおよびFe3+錯体/キサンチンオキシダーゼ系ー膜脂質過酸化反応の比較検討


低密度リポタンパクの過酸化により生じる血小板活性化因子様リン脂質

低密度リポタンパクの過酸化により生じる血小板活性化因子様リン脂質


ラット血漿のリゾホスファチジン酸生成康酵素活性

ラット血漿のリゾホスファチジン酸生成康酵素活性


LDLの過酸化により生成するPAF様脂質の分解

LDLの過酸化により生成するPAF様脂質の分解


Characterization of PAF-like phospholipids formed by lipid peroxidation

Journal of Lipid Mediators and Cell Signalling


保温血漿および卵胞液に存在するリゾホスファチジン酸の生成機構

保温血漿および卵胞液に存在するリゾホスファチジン酸の生成機構


リゾホスファチジン酸のラット培養血管平滑筋細胞増殖作用

リゾホスファチジン酸のラット培養血管平滑筋細胞増殖作用


脂質過酸化反応により派生するPAF様リン脂質の構造解析

脂質過酸化反応により派生するPAF様リン脂質の構造解析


アルキルエーテル型ホスファチジン酸の好中球における膜内動態

アルキルエーテル型ホスファチジン酸の好中球における膜内動態


ウシ脳組織中のPAF構造類似体:GC-MSによるα-ヒドロキシカルボン酸含有リン脂質の同定


Lipid peroxidation induced by hydroxyl-and lipid free-radicals and iron-oxygen complexes,and its inhibition by a-tocopherol

Medical,Biochemical and Chemical Aspects of Free Radicals


Increased platelet-activating factor (PAF) synthesis in polymorphonuclear leukocytes of vitamin E-deficient rats


脳組織中のPAF及び関連リン脂質類

脳組織中のPAF及び関連リン脂質類

Proceedings of the Japanese Conference on the Biochemistry of Lipids


PAFとそのリゾ体の血小板および好中球での膜内動態

PAFとそのリゾ体の血小板および好中球での膜内動態


[Fe2+錯体ー酸素]複合体によるリノール酸の過酸化誘導とα-トコフェロール(α-Toc)の抗酸化作用


ウシ脳組織中のPAF分子種の同定並びに定量

ウシ脳組織中のPAF分子種の同定並びに定量


血小板表面におけるエーテル結合型リン脂質の作用様式

血小板表面におけるエーテル結合型リン脂質の作用様式


Occurrence of new hypotensive and platelet-activating substances in bovine brain

Adv.Prostaglandins Thomboxane Leukotriene Res.


リゾホスファチジン酸のトリメチルシリル体のGC-MS分析

リゾホスファチジン酸のトリメチルシリル体のGC-MS分析


リポソーム膜におけるビタミンE同族体の脂質抗酸化作用

リポソーム膜におけるビタミンE同族体の脂質抗酸化作用


リポソーム膜における脂質過酸化反応とビタミンE

リポソーム膜における脂質過酸化反応とビタミンE


リゾホスファチジン酸及びその構造類似体の心血管作用

リゾホスファチジン酸及びその構造類似体の心血管作用


膜流動性および相転移に及ぼすトコフェロールの効果

膜流動性および相転移に及ぼすトコフェロールの効果


市販大豆レシチン中の血圧活性脂質の単離と同定

市販大豆レシチン中の血圧活性脂質の単離と同定


Presence of bioactive lysophosphatidic acid in renal effluent of rtas with unilateral ureteral obstruction

Abnormal production of lysophosphatidic acid (LPA), an important lysophospholipid mediator, in the kidney was examined to participate in the pathogenesis of renal fibrosis in rats. The secretory lysophospholipase D activity of autotoxin was considered as a possible pathway for extracellular production of LPA in the pathological renal fluids. In this study of rats with unilateral ureteral obstruction for two weeks, we measured concentrations of LPA and its precursor, lysophosphatidylcholine stored in the urinary bladder and present in the swollen pelvis of the ligated kidney as well as the corresponding blood plasma by liquid chromatography-tandem mass spectrometry. We found that concentrations of LPA and lysophosphatidylcholine accumulated in the effluent in the swollen pelvis of the ligated kidney of unilateral ureteral obstruction rats were much higher than those in the urinary bladder. The molecular species composition of LPA in the former was considerably different from that in the blood plasma, indicating the involvement of an additional source other than the blood circulation supplying LPA to the effluent in the swollen kidney. A potential mechanism is increased release of LPA from activated renal cells in the ureter-ligated kidney. Both pathways for supply of extracellular LPA may participate in the induction and progression of renal tubulofibrosis.